TWI823252B - System and method for continuous cell production - Google Patents

Abstract

The application provides a system for continuous cell production, comprising: a culture container; and a polymer blended layer, arranging on the inner surface of the culture container; wherein, the polymer blended layer is a pH responsive polymer blended with nylon. Additionally, a method for continuous cell production using the system of the present application is provided.

Description

Continuous cell production system and method

The present application relates to a method for utilizing a polymer blended layer in a continuous cell production system and its continuous cell production, in particular, blending nylon with an acid-base responsive polymer.

In the past decade or so, environmental pH-responsive polymer materials have been used in many applications in the field of biomaterials, including drug/gene release, tissue engineering, and separation procedures. However, due to the rise of environmental awareness, in addition to pursuing excellence in performance, developers have also returned to nature, hoping to find natural materials that can be sustainably reused while taking into account their performance.

Generally speaking, animal cells can be divided into three categories based on their growth patterns: suspension cells (such as blood cells, lymphoid tissue cells, hematopoietic stem cells), adherent cells (such as mesenchymal stem cells, epithelial cells) and mediator cells. Among the two types of cells, adherent cells need to be attached to an object in order to grow. Therefore, special treatment is required to separate them from the surface of the object. However, common processing procedures are often too complicated, or may damage the structure of cells during the separation process, causing their death and affecting subsequent research.

In this regard, the Republic of China Patent Publication No. I432577 discloses the use of acid-base responsive polymers (such as chitosan) as the substrate of a culture dish or coating on the culture dish, and by regulating the pH value of the cell culture medium to control cell attachment. Or detach, so that cells can be easily detached without using enzymes or extensive cleaning to avoid cell damage or death.

The Republic of China Patent Publication No. I558813 also discloses mixing acid-base responsive polymers (such as chitosan) and polymers (such as PCL) as the substrate of a culture dish or coating on the culture dish for cell culture. By controlling the pH value of the cell culture medium, in addition to controlling the attachment or detachment of cells, the efficiency (amount of cell attachment/detachment) and speed (cell attachment/detachment) can also be controlled based on the blending of different types of polymers and their respective proportions. attachment/detachment rate) to further achieve the effect of cell purification or sorting.

However, although previous technologies have solved the problem of detached cells in cell culture, in academic research or industrial utilization, there is a continuous need for sufficient numbers and good quality of cells. Therefore, after collecting the cells, they still need to be diluted, Re-seeding, re-cultivation, re-attachment and growth of cells and then detachment and collection again, repeated operations to achieve continuous performance of cell collection for use. However, the steps are cumbersome, and repeated attachment and detachment are not conducive to cell growth. At the same time, the effective expansion of stem cells during culture and the performance of native stemness are related to the quality of the cells. The re-implanted cells may also be unable to attach due to quality issues. , unable to expand or have poor activity, etc. Therefore, new cell culture technologies are still needed to provide effective and substantial benefits to academic research and industry in terms of cell quantity and quality.

In view of the problems existing in the prior art, the present application provides a continuous cell production system, including: a culture container; and a polymer mixed layer, which is disposed on the inner surface of the culture container; wherein the polymer mixed layer is made of acid Alkali-responsive polymer blended with nylon.

In one embodiment, the continuous cell production system includes a culture medium and/or a cell.

In one embodiment, the acid-base responsive polymer is selected from the group consisting of polymers with amine groups, chitosan, polylysine, polyacrylamine, polyacrylamide, polyhexamethylenediamine, and polypropylenediamine. , polybutylenediamine, polypentamethylenediamine and a group composed of their combinations.

In one embodiment, the weight ratio of the acid-base responsive polymer blended with nylon is 1:1 to 10:1.

In one embodiment, the polymer blended layer is prepared by uniformly mixing a solution of acid-base responsive polymer and nylon, coating it on the inner surface of the container and drying it. In another embodiment, the polymer blended layer has a plurality of nylon regions on the coating layer of the acid-base responsive polymer.

In addition, the present application also provides a method for continuous cell production, which includes: (1) providing a system as described in this application; (2) controlling the pH value of the culture medium in the range of 6.9-7.4 to culture the cells; and (3) ) Adjust the pH range of the culture medium to 7.5-8.5 to detach the cells.

In one embodiment, the aforementioned steps (2) and (3) are repeatedly performed. Preferably, the aforementioned steps (2) and (3) can be repeated for more than three times.

In one embodiment, the cell line is selected from stem cells, preferably adipose-derived stem cells, mesenchymal stem cells or skin stem cells; fibroblast cells, preferably foreskin fibroblasts Or embryonic fibroblasts; epithelial cells, preferably proximal renal tubule epithelial cells; cancer cells, preferably epithelial lung cancer cells; keratinocytes; corneal stromal cells and epidermal cells. group.

This application can preferably utilize a polymer blended layer of chitosan-nylon to establish a continuous cell production system. Since chitosan has acid-base response properties, it can control cell attachment and detachment by adjusting the pH value of the culture medium, but it is not good for cell growth. On the contrary, although nylon does not have acid-base response properties, it has good cell attachment and detachment. growth, making the culture medium acidic The base value can still allow cells to attach when the pH value is adjusted. When the pH value of the culture medium returns to control, the attached cells can continue to expand. The method of this application is to first allow cells to attach to this mixed material under appropriate pH control, and then adjust the pH value of the culture medium to allow chitosan to work to detach and collect the cells. After that, the cells are returned to the mixed material. The culture medium is controlled at the initial pH value, and the remaining cells continue to adhere to the nylon and continue to grow until the plate is full. The same action is then repeated to detach the cells and collect them. This cycle of operation is a continuous cell production system and method. .

Figure 1 shows the test results of system cultured cells using different materials.

Figure 2 shows the cell activity results of cells cultured in culture media extracted from different materials in each cycle.

Figure 3 shows the results of cells cultured on different single materials.

Figure 4 shows the cell activity results of cells cultured on different single materials.

Figure 5 shows the test results of cells cultured with different cells and mixed mixing methods.

The advantages and features of the invention, as well as the methods for achieving them, will be more readily understood when described in more detail with reference to exemplary embodiments and the accompanying drawings. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make this disclosure more thorough and complete and fully convey the scope of the invention to those of ordinary skill in the art, and the invention is only within the scope of the appended claims. defined. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be understood that terms such as those defined in commonly used dictionaries are to be understood to have meanings consistent with the context of the relevant art and are not to be overly idealistic or overly formal unless expressly defined herein. understand the meaning. As used throughout this specification, range values are intended to be shorthand representations of each and every value within the range, and any number within the range may be selected as the endpoint of the range.

The invention provides a continuous cell production system, which includes: a culture container; and a polymer mixed layer, which is arranged on the inner surface of the culture container; wherein the polymer mixed layer is an acid-base responsive polymer mixed with a resistant Lun.

In one embodiment, the continuous cell production system includes a culture medium and/or a cell.

In one embodiment, the acid-base responsive polymer is selected from the group consisting of polymers with amine groups, chitosan, polylysine, polyacrylamine, polyacrylamide, polyhexamethylenediamine, and polypropylenediamine. , polybutylenediamine, polypentamethylenediamine and a group composed of their combinations.

In one embodiment, the weight ratio of the acid-base responsive polymer blended with nylon is 1:1 to 10:1.

In one embodiment, the polymer blended layer is prepared by uniformly mixing a solution of acid-base responsive polymer and nylon, coating it on the inner surface of the container and drying it. In another embodiment, the polymer blended layer has a plurality of nylon regions on the coating layer of the acid-base responsive polymer.

In addition, this application also provides a method for continuous cell production, including: (1) providing a system as described in this application; (2) Control the pH value range of the culture medium to 6.9-7.4 to culture the cells; and (3) Adjust the pH value range of the culture medium to 7.5-8.5 to detach the cells.

In one embodiment, the aforementioned steps (2) and (3) are repeatedly performed. Preferably, the aforementioned steps (2) and (3) can be repeated for more than three times.

In one embodiment, the cell line is selected from stem cells, preferably adipose-derived stem cells, mesenchymal stem cells or skin stem cells; fibroblast cells, preferably foreskin fibroblasts Or embryonic fibroblasts; epithelial cells, preferably proximal renal tubule epithelial cells; cancer cells, preferably epithelial lung cancer cells; keratinocytes; corneal stromal cells and epidermal cells. group.

Several examples are listed below as examples to illustrate the implementation of the present application; those familiar with this art can easily understand the advantages and effects that can be achieved by this invention through the content of this description, and can carry out the work without departing from the spirit of this invention. Various modifications and changes to implement or apply the content of this creation.

Example 1 - Preparation of continuous cell production system

One aspect of this application is uniform mixing. First, mix acid-base responsive polymer (take chitosan as an example) and nylon (nylon-6,6) in a weight ratio of 1:1 to 10:1 (take a weight ratio of 5:1 as an example) Dissolve in the eutectic solvent (take formic acid or acetic acid as an example) and stir evenly at room temperature. Then, pour the prepared solution into a TCPS petri dish, dry it in an oven O/N at 60°C, neutralize it with 0.5N NaOH, rinse it with deionized water, and finally sterilize it under a UV lamp for one hour. be usable.

Another method is island-shaped mixing, which is to first apply the acid-base responsive polymer (take chitosan as an example) solution to the TCPS petri dish and dry it, and then drop the nylon solution at different positions. (The weight ratio of chitosan to nylon is 1:1 to 10:1), so that a plurality of island-shaped nylon areas are formed on the surface, which can be used after drying, neutralization, rinsing and sterilization.

Example 2 - Experiments on system culture of cells using different materials

This experiment uses 5 types of culture systems for continuous cell culture, which are:

1. CS(AA): Use acetic acid as the solvent and only use chitosan to coat the TCPS culture dish.

2. CS(FA): Use formic acid as the solvent and chitosan alone to coat the TCPS culture dish.

3. PCL/CS: Use acetic acid as a co-solvent, evenly mix polycaprolactone (PCL) and chitosan at a weight ratio of 1:5, and apply it to a TCPS culture dish.

4. NL/CS: Use formic acid as the solvent, mix nylon and chitosan evenly at a weight ratio of 1:5, and apply it to the TCPS culture dish.

5. GLTN/CS: Use acetic acid as a co-solvent, mix gelatin and chitosan evenly at a weight ratio of 1:5, and apply it to a TCPS culture dish.

This example uses the Hs68 cell line to conduct a continuous culture test. First, the cells are seeded in the above-mentioned culture system using a pH 6.99 medium and allowed to adhere and grow for 48 hours (expansion period). Then, the original medium is removed and a pH 7.65 medium is added. Maintain for 1 hour to allow the cells to complete detachment and collect (harvest period). The above operation is a culture cycle. Repeat the step of adding pH 6.99 culture medium for 48 hours and then adjusting to pH 7.65. Maintain for 1 hour and then collect the cells. 4 consecutive steps. cycle.

The test results are shown in Figure 1. Chitosan (CS) alone cannot achieve the purpose of continuous cell production at all. After the cells are detached in the first cycle, the cells cannot continue to grow and expand in the second cycle. It does not matter the choice of solvent (acetic acid or formic acid); on PCL/CS and GLTN/CS, although the pH value of the culture medium was increased in the first cycle, cells desorbed, but then continued to be cultured for 48 hours in the second cycle. The degree of cell growth and expansion has been obviously reduced a lot, and gradually decreases with the increase of cycle number, resulting in a state of sluggishness in the third cycle. It is almost completely unable to continue to grow; on the contrary, on NL/CS, the effects of cell growth/expansion and cell detachment can be clearly seen in each cycle, and can reach at least 3 to 4 cycles, showing that the use of acid Alkali-responsive polymers mixed with nylon can be used for continuous cell production. Unlike traditional cell culture methods, cells need to be diluted again after being collected and re-seeded for culture. Example 3 - Analysis of different materials for cell culture

Considering that during repeated operations, materials may dissolve and cause toxicity, which will cause some of the cells in the mixed material group to slump, so this experiment also collected the cells of each material in Example 2 after 48 hours of culture in the expansion phase of each cycle. Cell culture medium (extracted medium), also used the Hs68 cell line, seeded it into a 96-well TCPS dish at 3500 cells/well, added the medium extracted from each cycle above and cultured for 48 hours, collected the cells, and used MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide) assay was used to analyze cell viability. It can be seen from Figure 2 that this doubt can be eliminated. The activity of cells cultured in the culture medium extracted from different materials in each cycle is equivalent, and there is no decrease in activity due to possible dissolution of the materials to produce toxicity.

In addition, this experiment further explores the growth and activity of cells under a single material. In this experiment, the bottom of the TCPS plate was coated with a single material, polycaprolactone (PCL), nylon (NL) or gelatin (GLTN), and the Hs68 cell line was also planted. The cell growth was observed at 24 hours and 48 hours of culture. situation, and analyzed cell viability using MTT assay. As can be seen from Figure 3, the results of cell culture on the TCPS plate and those coated with different single materials are equivalent. No matter which material is used, the cells can adhere well and grow to fill the plate. At the same time, it can be seen from the cell activity analysis results in Figure 4 that there is no significant difference in the cell activity of cells cultured on each material. The activity of cells cultured on PCL is even slightly better than that of other materials. However, the PCL/CS hybrid The blending in Example 2 has proven that multiple cycles of cell culture cannot be continuously performed, indicating that the reason why NL/CS blending can be used for continuous cell production is not simply the characteristics of the nylon material itself on cells.

It can be seen from this example that the fact that cells can complete continuous cell production on the NL/CS polymer blended layer is not because nylon can improve the activity of cell culture, nor is it because the dissolution toxicity of other polymer materials reduces cell activity. Because nylon and acid-base-responsive polymers complement each other when mixed, other mixed polymer materials (such as PCL) used in other previous technologies can detach cells, but cannot be continuously cultured for multiple cycles.

Example 4 - Experiment on culturing cells with different cells and mixing methods

In this experiment, Hs68 (fibroblasts), adipose-derived stem cells (ADSC) and human renal proximal tubular epithelial cells (hRPTEC) were cultured on cells coated with NL/CS (1 :5) Evenly mixed culture dishes; In addition, Hs68 (fibroblasts) and human renal proximal tubular epithelial cells (hRPTEC) were cultured on a plate coated with NL/CS (1:5) Petri dish for island mixing. The culture conditions and methods are the same as in Example 2.

The test results are shown in Figure 5. Whether you are culturing Hs68, ADSC or hRPTEC, or using a uniform mixing or island mixing system, you can achieve very good growth/amplification and detoxification by adjusting the pH value of the culture medium. At the same time, the cell production is carried out in a continuous manner for at least 4 cycles. In summary, the invention technology and test results disclosed above, the continuous cell production system and method disclosed in this application can continuously and periodically drive multiple cell production cycles, providing very substantial help and benefits for academic research and industrial utilization.

Claims (10)

A continuous cell production system includes: a culture container; and a polymer blending layer arranged on the inner surface of the culture container; wherein the polymer blending layer is an acid-base responsive polymer blended with nylon. The system according to claim 1, which includes a culture medium and/or a cell. The system as described in claim 1, wherein the acid-base responsive polymer is selected from the group consisting of polymers with amine groups, chitosan, polylysine, polyacrylamine, polyacrylamide, and polyhexane. A group consisting of diamine, polypropylenediamine, polybutylenediamine, polypentylenediamine and their combinations. The system described in claim 1, wherein the weight of the acid-base responsive polymer blended with nylon is 1:1 to 10:1. The system as described in claim 1, wherein the polymer blended layer is obtained by uniformly mixing a solution of acid-base responsive polymer and nylon, coating it on the inner surface of the container and drying it. The system according to claim 1, wherein the polymer blended layer has a plurality of nylon regions on the coating layer of the acid-base responsive polymer. A method for continuous cell production, comprising: (1) providing the system as described in claim 2; (2) controlling the pH value of the culture medium in the range of 6.9-7.4 to culture the cells; and (3) adjusting the The pH value of the culture medium ranges to 7.5-8.5 to allow the cells to detach. The method described in claim 7, wherein step (2) and step (3) are repeatedly performed. The method described in claim 8, wherein step (2) and step (3) can be repeated more than three times. The system as described in claim 2, wherein the cell line is selected from stem cells, preferably adipose-derived stem cells, mesenchymal stem cells or skin stem cells; fibroblast cells, preferably The ground is foreskin fibroblasts or embryonic fibroblasts; epithelial cells, preferably proximal renal tubule epithelial cells; cancer cells, preferably epithelial lung cancer cells; keratinocytes; corneal stromal cells and A group of epidermal cells.